Neurofilament gene expression in transgenic mice

1. DNA fragments that include the human neurofilament NF-L gene was found to be correctly expressed in the majority of neurons in transgenic mice. 2. The NF-L transgene product, which is detectable in situ with a species-specific monoclonal antibody, provides a powerful genotype marking system applicable to developmental and regeneration studies of the mammalian nervous system. 3. The proximal 5'-flanking region of the NF-L gene is sufficient to direct expression of a heterologous gene in the mouse nervous system.     

A new variant of Charcot-Marie-Tooth disease type 2 is probably the result of a mutation in the neurofilament-light gene

Charcot-Marie-Tooth (CMT) disease is the most common inherited motor and sensory neuropathy. The axonal form of the disease is designated as "CMT type 2" (CMT2). Although four loci known to be implicated in autosomal dominant CMT2 have been mapped thus far (on 1p35-p36, 3q13. 1, 3q13-q22, and 7p14), no one causative gene is yet known. A large Russian family with CMT2 was found in the Mordovian Republic (Russia). Affected members had the typical CMT2 phenotype. Additionally, several patients suffered from hyperkeratosis, although the association, if any, between the two disorders is not clear. Linkage with the CMT loci already known (CMT1A, CMT1B, CMT2A, CMT2B, CMT2D, and a number of other CMT-related loci) was excluded. Genomewide screening pinpointed the disease locus in this family to chromosome 8p21, within a 16-cM interval between markers D8S136 and D8S1769. A maximum two-point LOD score of 5.93 was yielded by a microsatellite from the 5' region of the neurofilament-light gene (NF-L). Neurofilament proteins play an important role in axonal structure and are implicated in several neuronal disorders. Screening of affected family members for mutations in the NF-L gene and in the tightly linked neurofilament-medium gene (NF-M) revealed the only DNA alteration linked with the disease: a A998C transversion in the first exon of NF-L, which converts a conserved Gln333 amino acid to proline. This alteration was not found in 180 normal chromosomes. Twenty unrelated CMT2 patients, as well as 26 others with an undetermined form of CMT, also were screened for mutations in NF-L, but no additional mutations were found. It is suggested that Gln333Pro represents a rare disease-causing mutation, which results in the CMT2 phenotype.     

Structure and evolutionary origin of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein

We describe the complete sequence of the gene encoding mouse NF-M, the middle-molecular-mass neurofilament protein. The coding sequence is interrupted by two intervening sequences which align perfectly with the first two intervening sequences in the gene encoding NF-L (the low-molecular-mass neurofilament protein); there is no intron in the gene encoding NF-M corresponding to the third intron in NF-L. Therefore, both the number of introns and their arrangement in the genes coding NF-L and NF-M contrast sharply with the number and arrangement of introns in the genes of known sequence, encoding other members of the intermediate filament multigene family (desmin, vimentin, glial fibrillary acidic protein and the acidic and basic keratins); with the exception of a single truncated keratin gene that lacks an encoded tailpiece, these genes all contain eight introns, of which at least six are placed at homologous locations. Assuming the existence of a primordial intermediate filament gene containing most (if not all) the introns found in contemporary non-neurofilament intermediate filament genes, it seems likely that an RNA-mediated transposition event was involved in the generation of an ancestral gene encoding the NF polypeptides. A combination of insertional transposition and gene-duplication events could then explain the anomalous number and placement of introns within these genes. Consistent with this notion, we show that the genes encoding NF-M and NF-L are linked.     

Effect of phosphorylation on 68 KDa neurofilament subunit protein assembly by the cyclic AMP dependent protein kinase in vitro

The effect of phosphorylation by cyclic AMP dependent protein kinase on the assembly of the core-forming 68 KDa neurofilament subunit protein (NF-L) was studied in vitro by fluorescence energy transfer and electron microscopy. Phosphorylation of unassembled NF-L in a low ionic strength buffer by cyclic AMP dependent protein kinase led to the incorporation of 1-2 phosphate groups/mole protein. Assembly of this phosphorylated NF-L was inhibited significantly; compared to non-phosphorylated NF-L, the critical concentration of phosphorylated NF-L was raised by greater than 30-fold. Assembled NF-L filaments could also be phosphorylated by cyclic AMP dependent protein kinase indicating that the sites were accessible. Phosphorylation of NF-L in the filamentous state induced their disassembly. The results suggest that phosphorylation by cyclic AMP dependent protein kinase is a possible means to modulate the assembly state of NF-L.     

Interleukin-1beta up-regulates expression of neurofilament light in human neuronal cells

Elevated expression of interleukin-1 (IL-1beta), a pro-inflammatory cytokine secreted by activated microglia, is a pathogenic marker of numerous neurodegenerative processes including Alzheimer's disease (AD). We have characterized a link between IL-1beta and the 68-kDa neurofilament light (NF-L) protein, which is a major component of the neuronal cytoskeleton. Using human brain aggregate cultures, we found that IL-1beta treatment significantly increased NF-L expression in primary neurons. Analysis of mRNA levels demonstrated elevated NF-L expression within 72 h while imaging of neurons by immunofluorescent staining for NF-L confirmed IL-1beta-induced NF-L protein expression. These observations suggest a potential inflammatory-induced mechanism for deregulation of an important cytoskeletal protein, NF-L, possibly leading to neuronal dysfunction.     

Neurofilament deficiency in quail caused by nonsense mutation in neurofilament-L gene

The existence of a neurofilament-deficient mutant of Japanese quail was recently documented (Yamasaki, H., C. Itakura, and M. Mizutani. 1991. Acta Neuropathol. 82:427-434), but the genetic events leading to the neurofilament deficiency have yet to be determined. Our molecular biological analyses revealed that the expression of neurofilament-L (NF- L) gene was specifically repressed in neurons of this mutant. To search for mutation(s) responsible for the shutdown of this gene expression, we cloned and sequenced the NF-L genes in the wild-type and mutant quails. It is eventually found that the NF-L gene in the mutant includes a nonsense mutation at the deduced amino acid residue 114, indicating that the mutant is incapable of producing even a trace amount of polymerization-competent NF-L protein at any situation. The identification of this nonsense mutation provides us with a solid basis on which molecular mechanisms underlying the alteration in the neuronal cytoskeletal architecture in the mutant should be interpreted.     

Effects of phosphorylation of the neurofilament L protein on filamentous structures.

Effects of phosphorylation of the neurofilament L protein (NF-L) on the reassembly system were studied by both sedimentation experiments and low-angle rotary shadowing. Bovine spinal cord NF-L was phosphorylated with 3-4 mol/mol protein by either the catalytic subunit of cAMP-dependent protein kinase or protein kinase C. Phosphorylated NF-L could not assemble into filaments. Phosphorylation by either cAMP-dependent protein kinase or protein kinase C inhibited the same step of the reassembly process. Phosphorylated NF-L remained as an 8-chain complex even in favorable conditions for reassembly. The extent of the effect of phosphorylation on the filamentous structure of NF-L was also investigated by using the catalytic subunit of cAMP-dependent protein kinase. The amount of unassembled NF-L increased linearly with increased phosphorylation in the sedimentation experiments. Structural observations indicated that 1 or 2 mol of phosphorylation is enough to inhibit reassembly and to induce disassembly, and the disassembly process was also observed. The filaments were shown to unravel with disassembly. Star-like clusters, which we reported as being the initial stage of reassembly, were also identified.     

Binding of brain spectrin to the 70-kDa neurofilament subunit protein

Brain spectrin, or fodrin, a major protein of the subaxolemmal cytoskeleton, associates specifically in in vitro assays with the 70-kDa neurofilament subunit (NF-L) and with glial filaments from pig spinal cord. As an initial approach to the identification of the fodrin-binding proteins, a crude preparation of neurofilaments was resolved by electrophoresis on SDS/polyacrylamide gels and then transferred to nitrocellulose paper, which was 'blotted' with 125I-fodrin. A significant binding of fodrin was observed on polypeptides of 70 kDa, 52 kDa and 20 kDa. These polypeptides were further purified and identified respectively as the NF-L subunit of neurofilaments, the glial fibrillary acidic protein (GFP) and the myelin basic protein. The binding of fodrin to NF-L was reversible and concentration-dependent. The ability of the pure NF-L and GFP to form filaments was used to quantify their association with fodrin. a) The binding of fodrin to reassembled NF-L was saturable with a stoichiometry of 1 mol fodrin bound/50 +/- 10 mol NF-L and an apparent dissociation constant Kd = 4.3 x 10(-7) M. b) The binding involved the N-terminal domain of the polypeptide chain derived from the [2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine] cleavage of NF-L. c) Binding occurred optimally at physiological pH (6.8-7.2) and salt concentrations (50 mM). d) Interestingly, calmodulin, a Ca2+-binding protein, which has been shown to bind to fodrin, was found to reinforce the binding of fodrin to the NF-L, at Ca2+ physiological concentrations. The binding of fodrin to pure neurofilaments was not affected by the presence of the 200-kDa (NF-H) and the 160-kDa (NF-M) subunits. The apparent dissociation constant for the binding of fodrin to NF-L in the pure NF was 1.0 x 10(-6) M with 1 mol fodrin bound/80 +/- 10 mol NF-L. Moreover, the binding of fodrin to GFP, demonstrated in blot assays, was confirmed by cosedimentation experiments. The apparent dissociation constant Kd for the fodrin binding was 2.8 x 10(-7) M and the maximum binding was 1 mol fodrin/55 +/- 10 mol GFP.     

Isolation of a stable tetramer of the low molecular weight component of neurofilaments (NF-L)

When crude neurofilaments were dissolved in a solution containing 8 M urea and 1% beta-mercaptoethanol (beta-ME), the component proteins of the neurofilaments and other contaminating filaments were solubilized into monomeric forms. However, when reassembled filaments were solubilized again by the addition of urea to 8 M without beta-ME, several bands which seemed to be oligomeric forms of filament proteins were observed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among them, a band which appeared between microtubule-associated protein-1 (MAP-1) and fodrin was most remarkable. This band was also observed when a triplet mixture of the neurofilaments (NF-H, NF-M, NF-L) was reassembled. The molecular weight of this band was estimated to be 280 kDa. In addition, much of this component was easily isolated on DE-52 column chromatography of the reassembled crude neurofilament proteins with buffers containing 6 M urea, while the low molecular weight component of the neurofilaments (NF-L, 70 kDa) was hardly detected. Furthermore, the isolated 280 kDa component was reduced to NF-L on the addition of beta-ME to 1%. In contrast, the 280 kDa component was produced on dialysis of isolated NF-L against the assembly buffer. From these results, it is deduced that this component is the stable tetramer of NF-L which is produced through spontaneous interchain disulfide formation among protofilament tetramers.     

Characterization of NF-L and betaIISigma1-spectrin interaction in live cells.

Neurofilaments (NFs) are neuron-specific intermediate filaments (IFs) composed of three different subunits, NF-L, NF-M, and NF-H. NFs move down the axon with the slow component of axonal transport, together with microtubules, microfilaments, and alphaII/betaII-spectrin (nonerythroid spectrin or fodrin). It has been shown that alphaII/betaII-spectrin is closely associated with NFs in vivo and that betaII-spectrin subunit binds to NF-L filaments in vitro. In the present study we seek to elucidate the relationship between NF-L and betaII-spectrin in vivo. We transiently transfected full-length NF-L and carboxyl-terminal deleted NF-L mutants in SW13 Cl.2 Vim- cells, which lack an endogenous IF network and express alphaII/betaIISigma1-spectrin. Double-immunofluorescence and electron microscopy studies showed that a large portion of betaIISigma1-spectrin colocalizes with the structures formed by NF-L proteins. We found a similar association between NF-L proteins and actin. However, coimmunoprecipitation experiments in transfected cells and the yeast two-hybrid system results failed to demonstrate a direct interaction of NF-L with betaIISigma1-spectrin in vivo. The presence of another protein that acts as a bridge between the membrane skeleton and neurofilaments or modulating their association may therefore be required.     

Site-specific phosphorylation of neurofilament-L is mediated by calcium/calmodulin-dependent protein kinase II in the apical dendrites during long-term potentiation

Neurofilament-L (NF-L), one subunit of the neuronal intermediate filaments, is a major element of neuronal cytoskeletons. The dynamics of NF-L are regulated by phosphorylation of its head domain. The phosphorylation sites of the NF-L head domain by protein kinase A, protein kinase C, and Rho-associated kinase have been previously identified, and those by calcium/calmodulin-dependent protein kinase II (CaMKII) were identified in this study. A series of site- and phosphorylation state-specific antibodies against NF-L was prepared to investigate NF-L phosphorylation in neuronal systems. Long-term potentiation (LTP) is a cellular model of neuronal plasticity that is thought to involve the phosphorylation of various proteins. NF-L is considered a possible substrate for phosphorylation. During LTP stimulation of mouse hippocampal slices, the series of antibodies demonstrated the increase in the phosphorylation level of Ser(57) in NF-L and the visualization of the localized distribution of Ser(57) phosphorylation in a subpopulation of apical dendrites of the pyramidal neurons. Furthermore, Ser(57) phosphorylation during LTP is suggested to be mediated by CaMKII. Here we show that NF-L is phosphorylated by CaMKII in a subpopulation of apical dendrites during LTP, indicating that Ser(57) is a novel phosphorylation site of NF-L in vivo related to the neuronal signal transduction.     

Neurofilament-light increases the cell surface expression of the N-methyl-D-aspartate receptor and prevents its ubiquitination

NMDA (N-methyl-D-aspartate) subtype of glutamate receptors are core components of dendritic spine postsynaptic densities (PSDs), in which they are anchored via their carboxy-terminal tails to cytoskeletal proteins. In this study, we examined the role of the neuronal intermediate filament protein, neurofilament-light (NF-L), also a component of the PSD, in the regulation of NMDA receptor (NMDAR) expression and function in a heterologous system. Coexpression of NF-L with NR1 or NR2B subunits of the NMDAR in HEK293 (human embryonic kidney 293) cells did not result in surface expression as measured by surface biotinylation and cell ELISAs, whereas the combined expression of the three elements resulted in a 20% increase in the surface abundance of NR1, along with a concomitant increase in NMDAR-mediated cytotoxicity. Investigating the origin of this increase, we found that the NR1 subunits are ubiquitinated in HEK293 cells, and that the coexpression of NF-L antagonizes this process. These results suggest a possible means of stabilization of NR1 via its association with NF-L.